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Effect of Antioxidant Supplementation on the Sperm Proteome of Idiopathic Infertile Men

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Effect of Antioxidant Supplementation on the Sperm Proteome of Idiopathic Infertile Men

Ashok Agarwal et al. Antioxidants (Basel).

Abstract

Antioxidant supplementation in idiopathic male infertility has a beneficial effect on semen parameters. However, the molecular mechanism behind this effect has not been reported. The objective of this study was to evaluate the sperm proteome of idiopathic infertile men pre- and post-antioxidant supplementation. Idiopathic infertile men were provided with oral antioxidant supplementation once daily for a period of 6 months. Of the 379 differentially expressed proteins (DEPs) between pre- and post-antioxidant treatment patients, the majority of the proteins (n = 274) were overexpressed following antioxidant treatment. Bioinformatic analysis revealed the activation of oxidative phosphorylation pathway and upregulation of key proteins involved in spermatogenesis, sperm maturation, binding of sperm, fertilization and normal reproductive function. In addition, the transcriptional factors associated with antioxidant defense system (PPARGC1A) and free radical scavenging (NFE2L2) were predicted to be functionally activated post-treatment. Key DEPs, namely, NDUFS1, CCT3, PRKARA1 and SPA17 validated by Western blot showed significant overexpression post-treatment. Our novel proteomic findings suggest that antioxidant supplementation in idiopathic infertile men improves sperm function at the molecular level by modulating proteins involved in CREM signaling, mitochondrial function and protein oxidation. Further, activation of TRiC complex helped in nuclear compaction, maintenance of telomere length, flagella function, and expression of zona pellucida receptors for sperm-oocyte interaction.

Keywords: antioxidants; bioinformatics; idiopathic; male infertility; sperm proteome.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
(a) A schematic representation of the overall experimental design that was used to identify the proteomic changes associated with antioxidant (‘FH PRO for men’) treatment in idiopathic infertile patients. N1 and N2 represent individual patient samples and the pooled sample is from three different individuals normalized for sperm concentration. Each sample was run in triplicate on one-dimensional SDS-PAGE gels and subjected to in-gel trypsin digestion. The soluble proteins were extracted, reduced, alkylated, and analyzed using liquid chromatography-tandem mass spectrometry. (b) Histogram showing homogeneity in the number of proteins identified in individual samples run in triplicate. G1N1 1-3: Gel-1 N1 (replicate 1-3); G1N2 1-3: Gel-1 N2 (replicate 1-3); G1N3 1-3: Gel-1 pooled sample (replicate 1-3); G2N1 1-3: Gel-2 N1 (replicate 1-3); G2N2 1-3: Gel-2 N2 (replicate 1-3); G2N3 1-3: Gel-2 pooled sample (replicate 1-3).
Figure 2
Figure 2
The number of sperm proteins identified in pre-and post-antioxidant treatment groups. Semen sample from 5 patients were used for the analysis in which two were individual sample while the third was a pooled sample from three different individuals.
Figure 3
Figure 3
(a) The number and (b) abundance of differentially expressed proteins identified in spermatozoa of idiopathic infertile patients pre-and post-antioxidant treatment.
Figure 4
Figure 4
Top ten enriched canonical pathways in idiopathic infertile men post-antioxidant treatment.
Figure 5
Figure 5
Predicted transcription factors PPARGC1A, NFE2L2 and HSF2 regulating the DEPs in idiopathic infertile men post-antioxidant treatment.
Figure 6
Figure 6
Activation of CREM signaling pathway in spermatozoa of idiopathic infertile patients post-antioxidant treatment.
Figure 7
Figure 7
Inhibition of the oxidation of protein pathway in spermatozoa of idiopathic infertile patients post-antioxidant treatment.
Figure 8
Figure 8
Expression profile of differentially expressed sperm proteins (Western blot) in spermatozoa of idiopathic infertile patients pre- (n = 8) and post-antioxidant treatment (n = 8). (a) NDUFS1, (b) CCT3, (c) PRKAR1A and (d) SPA17.

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